This project will use a team-based approach to pursue three directly interrelated areas of investigation important for learning to make new pancreatic beta cells from other cell types. In Aim 1 we seek to gain a deeper understanding of the molecular events that dictate formation of multiple endocrine cell types during pancreas development. This will be achieved by isolating a series of highly purified progenitor cell populations, performing digital gene expression analysis, and developing bioinformatics strategies for characterizing the differences among discrete cellular populations that will be temporally or genetically informative. In Aim 2 we will seek to utilize the knowledge gained in Aim 1 to determine the veracity of protocols being used to direct the differentiation of hESCs towards pancreatic cell fates and to rationally improve these protocols by monitoring the expression of gene clusters that are specifically activated or repressed during mouse development. In Aim 3 we will develop new mouse lines in which the expression of three transcription factors that have been reported to be capable of transdifferentiating pancreatic acinar to beta cells can be easily modulated by the administration of doxycycline. These mice will be used to explore how transdifferentiation actually occurs and to determine the extent to which the newly generated beta cells are functional and exhibit a gene expression profile similar to authentic beta cells. This project is based on the premise/hypothesis that multiple gene regulatory networks, which are normally set up during mouse development, must be established during directed or trans-differentiation of other cell types to achieve the beta cell-like functionalities necessary for clinical use. Greater knowledge of pancreas-specific gene regulatory networks, how they are established and differ among related cell populations, and determining whether they are present or absent in experimentally-derived cellular populations, will serve as a platform both for new discovery and protocol improvements. The six investigators in this project have a track record of productive collaborative interactions and bring specific knowledge and abilities necessary to accomplish these goals. Thus, it is anticipated that this project will generate important resources that will not only advance two of the overarching goals of the Beta Cell Biology Consortium but also have broad scientific impact and utility.